1. Field of the Invention
The present invention relates to an enzyme electrode and a method for producing the enzyme electrode.
2. Description of Related Art
Carbohydrate analysis can be performed by methods including optical rotation (Beychok and Kabat, 1965), colorimetry (Thomas and Dutcher, 1924), enzyme electrode (Qiu et al., 2009; Sun et al., 2008; Zhang et al., 2005), and high-performance liquid chromatography (HPLC) with various kinds of detector (Cai et al., 2005; Cheng and Chang, 2007; Cheng, et al., 2006; Kauppila et al., 2008; Qian et al., 2008; Raessler et al., 2008). Scientists used HPLC most for the simultaneous determination of several carbohydrates with good accuracy, precision, and sensitivity. However, in spite of the advantages of HPLC, the instrument for HPLC is bulky and expensive and the analysis time is usually lengthy (Cheng et al., 2010). Therefore, for many clinical applications such as the glucose monitoring for diabetes HPLC is not an ideal method to use. In recent years, the development of enzyme electrode method is seen to be a better technique for specific carbohydrate analysis by its advantages such as substrate specificity, easy operation, low cost, portable instrumentation, and faster analysis over the HPLC method (Qian et al., 2008).
Clark and Lyons developed the first generation of glucose enzyme electrode, and Schlapfer improved it by replacing oxygen with artificial mediator for eliminating the dependence of oxygen in the air. (Wang, 2008). Since then, ferrocene and its derivatives were used extensively as the artificial redox mediator (Escorcia and Dhirani, 2007; Qian et al., 2008; Wang and Du, 2004) for glucose enzyme electrode with their various desirable properties, e.g., a relatively low molecular mass, reversibility, regeneration at low potential, and generation of stable redox forms (Fernández and Carrero, 2005). Typically, the mediator is dissolved in an electrolyte for glucose analysis. However, ferrocene and its derivatives are carcinogenic and this property makes them not suitable for a lot of applications provided that they are disposed freely to the environment. One simple method to overcome this drawback is its immobilization to the electrode. Qiu and co-workers (2009) have reported an amperometric sensor from ferrocene carboxaldehyde directly and physically adsorbed on multiwalled carbon nanotubes for the determination of glucose. Reagentless enzyme-based glassy carbon electrode biosensor with a ferrocene-branched chitosan matrix for glucose analysis was also developed by Sun and co-workers (Yang et al., 2007).
Although many methods such as physical adsorption, cross-linking, and encapsulation have been disclosed to improve the immobilization of enzyme and even mediator to the electrode, so far it is still needed to provide an enzyme electrode with excellent accuracy, wide detectable concentration range, good stability, and long-term reusable capability.
In another aspect, the technique of flow injection analysis (FIA) was developed in 1976 by Denmark's scientists Ruzicka and Hansen (Zhi, 1998). Due to the advantages of fast continuous analysis, simplicity, and low cost, this technique is proved to be highly useful for practical applications in many fields, e.g., environmental monitoring, process analytical chemistry or clinical diagnostics (Thomaidis and Georgiou, 1999; Wang and Chen, 1995). FIA can be carried out either off-line or on-line mode; however, off-line FIA is usually tedious and may cause sample loss during sample transfer (Cheng and Chang, 2007). On-line FIA is thus used to solve these problems and to show the in-time monitoring ability. In recent years, the on-line FIA monitoring of glucose with biosensor in biological system (Kumar et al., 2001; Nandakumar et al., 1999) was usually coupled with microdialysis for sampling (Gramsbergen et al., 2004; Rhemrev-Boom et al., 2001; Yao et al., 2004). Accordingly, a need is arisen in the art to develop other types of sampling device coupled to on-line FIA that are more convenient to use.